Field of the Invention
The invention in the field of biochemistry, molecular biology and medicine relates to the treatment of severe lung inflammation and acute lung injury (ALI) by inhibition of Bruton's tyrosine kinase (Btk) in the lung using small inhibitory RNA (siRNA) that silences Btk expression or small molecule inhibitors of Btk.
Description of the Background Art
Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a life threatening inflammatory disease with mortality of 30 to 50%. One of the characteristic features of ALI/ARDS is a significant increase in the migration of neutrophils to lungs (18, 32, 34, 53). Multiple studies indicate a correlation between the number of neutrophils in the alveolar spaces and the resulting severity of the disease. Even though ARDS can develop in neutropenic patients, these individuals constitute a distinct minority of all ARDS cases, and, moreover, neutrophils contribute to the deterioration of lung function in patients recovering from neutropenia (4, 42). In this regard, K. Y. Yang et al. found that the early activation status of neutrophils in patients with ALI determines the clinical course of the disease (57). Many animal models of ALI are linked to presence of elevated concentrations of neutrophils (1, 11, 19, 49). Together these observations suggested to the present inventor that neutrophils may play a central role in the pathogenesis of most cases of clinical ALI/ARDS.
Both neutrophil apoptosis and the phagocytic uptake of apoptotic neutrophils (efferocytosis) are decreased at sites of pulmonary inflammation in ALI/ARDS. Impaired clearance of activated neutrophils in lungs of patients with ALI/ARDS leads to excessive accumulation of these cells at the site of inflammation and may promote lung injury (33, 35, 36).
Previous studies from the present inventor's laboratory presented a novel concept in the field of human ALI/ARDS, i.e., that Btk-associated pathways may play an important role in the pathophysiology of ALI/ARDS by influencing local inflammation (25). Btk, a Tec kinase, belongs to a family of non-receptor intracellular tyrosine kinases (38) which includes the structurally homologous kinases Btk, Tec, Itk, Bmx, and Txk. Tec kinases typically reside in an inactive form in the cytoplasm, and are translocated to the membrane fraction upon cell stimulation where they initiate down-stream signaling cascades (48).
The inactive form of Btk resides in the cytoplasm. Once activated, Btk typically migrates to the cell membrane (25, 48). Btk has been primarily studied in B lymphocytes where the engagement of the B cell receptor leads to its phosphorylation (7, 22). In human neutrophils Btk mediates signaling via toll-like receptor-4 (TLR4 receptor) and G-protein-coupled receptor (16, 55). Moreover, recent studies have shown that engagement of FcγRIIa receptors by immune complexes can also trigger Btk activation in these cells (25).
The present inventor and colleagues also noted (25) that there is cross talk between FcγRIIa and TLR4 in alveolar neutrophils from patients with ALI/ARDS and that Btk mediates the molecular cooperation between these two receptors. To study cross talk between TLR4 and FcγRIII (mouse equivalent of human FcγRIIa; 5, 15, 41) in vivo, they developed a unique two hit model of ALI (lipopolysaccharide (LPS)/immune complex (IC)-induced ALI). LPS was used because sepsis is a major risk factor for development of ALI/ARDS (34). In addition, since the previous studies showed that anti-KC:KC ICs (KC is an abbreviation for C-X-C motif ligand 1 or CXCL1) contribute in a significant way to severe lung inflammation in LPS treated mice (28), the inventor also employed anti-KC antibody:KC (anti-KC:KC) ICs. These studies with animal models of ALI as well as clinical samples from ALI/ARDS patients indicate that LPS dependent signaling induces a significant increase in the level of ICs in the alveolar compartment. Therefore a model was chosen that involves the combined effects of LPS and immune complexes to closely mimic the situation in patients with ALI. In summary, this model reflects very well the sequence of the pro-inflammatory events in patients with ALI, where the initial insult (such as LPS) triggers production of pathogenic ICs (3, 14, 26-30).
Infection with the influenza (“Flu”) virus triggers the rapid recruitment of neutrophils to the alveolar compartment, where these cells play an important role in host defense by controlling viral replication and clearing dying cells. Mounting evidence supports the contribution of neutrophils to the excessive acute inflammatory responses that cause severe lung immunopathology during Flu infection (D. Damjanovic et al., Clin. Immunol. 144:57-69, 2012; M N Ballinger et al. J. Interferon Cytokine Res. 30:643-652, 2010; B. Amulic et al. Annu. Rev. Immunol. 30:459-89, 2012).
In view of the present inventors' recent discoveries that engagement of FcγRIIa receptors by immune complexes can trigger activation of Btk in neutrophils (25) and their discovery disclosed herein that silencing Btk in neutrophils in lungs had a dramatic protective effect on in LPS/immune complex-induced ALI studies were directed to the role of Btk and the therapeutic effect of its silencing in influenza A induced ALI and in a mouse model of emphysema/chronic obstructive pulmonary disease (COPD).
COPD is the fourth leading cause of death worldwide. The inflammatory response to cigarette smoke appears to be the major etiological factor in the pathogenesis of COPD and exposure to second hand smoke (SHS) activates an inflammatory cascade in the lungs. At present, casual interventions that can stop progression of COPD are not available primarily because of the lack of thorough understanding of the mechanism underlying the development and natural course of this disease. Indeed specific pathways/mediators that drive the induction and progression of chronic inflammation, emphysema and altered lung function are not known. Therefore, there is a clear need in the art for new therapies that can prevent the induction and progression of COPD. A hallmark of COPD is a substantially enhanced inflammatory/immune response in the airways and lung, and COPD can be described as a chronic pulmonary disease characterized by progressive airflow limitation. The natural history of COPD typically begins with inflammatory changes in the larger airways (chronic bronchitis). Additional well-recognized features of COPD include remodeling and narrowing of the small airways and parenchymal tissue destruction with airspace enlargement (emphysema). Current treatments do not effectively inhibit chronic inflammation or reverse the pathology of COPD nor do they successfully target the factors that initiate and drive the long-term progression of the disease. Development of novel therapies requires animal models that adequately reflect pathophysiology of this disease. Animal models utilizing cigarette smoke exposure display the characteristic features of human COPD including the accumulation of macrophages, influx of neutrophils and T lymphocytes, increased release of pro-inflammatory mediators (cytokines, chemokines, proteases, and reactive oxygen species), small airway fibrosis/remodeling, mucus hypersecretion, lung dysfunction and the development of emphysema. Indeed exposure of mice to SHS remains the best animal system for defining, testing, and evaluating novel drug targets for COPD (P. Barnes, COPD 1:59-70, 2004; R L Birru et al. Front Physiol. 3:348, 2012; M. Podowski et al. J. Clin. Invest. 122:229-40, 2012; K. Pappas et al., Cytokine. 64:613-25, 2013; R. Vlahos et al., Clin. Sci. 126:253-65, 2014). After 20 weeks of smoke exposure, wild type (WT) mice display chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function which are characteristic features of COPD (E L Beckett et al., J. Allergy Clin. Immunol. 131:752-62, 2013). Vascular abnormalities are well known COPD comorbidities and include endothelial dysfunction, arterial stiffness and atherogenesis. Smokers suffer from both decline in lung function and cardiovascular problems. Recent studies demonstrated that vascular inflammation, endothelial dysfunction and oxidative modification of lipids may contribute to pathogenesis. Therefore, it is not surprising that abnormal lung morphology and substantial decrease in function are found in apolipoprotein E-deficient (ApoE−/− mice which are susceptible to cardiovascular issues and atherosclerosis. Exposure of such mice to cigarette smoke causes premature emphysema, abnormal lung inflammation, and airspace enlargement with altered mechanical properties in the lungs (G. Arunachalam et al., J. Inflamm. 7:34, 2010).
Zafra, M P et al. PLoS One 9(3): e91996, 2014, disclosed the silencing of a “suppressor of cytokine signaling” (SOCS3) in a mouse model of chronic asthma using siRNA delivered intranasally. Improvement in the eosinophil count and the normalization of hyperresponsiveness to methacholine were observed as were an improvement in mucus secretion and a reduction in lung collagen, said to be prominent features of airway remodeling. The results were said to imply involvement of the JAK/STAT and RhoA/Rho-kinase signaling pathways. The reference did not relate to targeted delivery to neutrophils, targeting of the Btk gene, nor treatment of acute lung injury.
Perl, M. et al. (Mol Med. 14:465-75. 2008) is a review discussing pathogenesis of ALI and describing use of siRNA in vivo to inhibit ALI. This document elucidates mechanisms of ALI pathogenesis focusing on two main theories: that neutrophils can play a central role in driving ALI and that lung epithelial cell apoptosis is an important pathogenic factor. The authors discuss a double-hit mouse model of indirect ALI induced by hemorrhagic shock (HEM) and subsequent polymicrobial sepsis. A strategy of using siRNA in mouse lungs in vivo is described (for understanding ALI pathology). Results from the HEM-induced septic ALI model implied that the tissue environment (infectious versus inflammatory) encountered by neutrophils is important in determining whether or not they mediate organ damage. Use of silencing RNA is said to represent a potentially powerful experimental approach to allow better understanding of the pathology of ALI and a therapeutic approach to treatment. The history of siRNA, its discovery, development, the mechanisms involved, as well as its successful initial uses in mammals in vivo are described in cited references (Kumar L D et al., Adv. Drug Deliv. Rev. 59:87-100, 2007; Aigner A., Curr. Opin. Mol. Ther. 9:345-52, 2007; de Fougerolles, A, et al., Nat. Rev. Drug Discov. 6:443-53, 2007; Martin S E et al., Annu. Rev. Genomics Hum. Genet. 8:81-108, 2007). The lung is said to be a good candidate for application in vivo, as it can be accessed straightforwardly by intranasal (i.n.) or intratracheal (i.t.) routes. Although nucleic acid transfer efficiency is known to be diminished substantially by the phospholipids and proteins of the airway surface liquid, unlike systemic delivery, the delivery of naked siRNA into lungs was efficient, potentially obviating a need for complex and costly approaches using vector systems or chemical siRNA modifications. The feasibility of a surfactant-based or naked siRNA approach in the mouse lung targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or heme oxygenase-1 during ischemia reperfusion, respectively, was noted. The document states that Phase I/II trials for Respiratory Syncytial Virus infection have been conducted and are on their way to evaluate safety, tolerability, and antiviral activity of siRNA treatment in human lungs. The authors described their initial attempts to extend their observation that blockade of MIP-2 or keratinocyte-derived chemokine (KC) with conventional antibodies effected the development of ALI (Lomas J L et al., Shock. 19:358-65, 2003) by silencing these chemokines locally in the lung (Lomas-Neira J L et al., J. Leukoc. Biol. 77:846-53, 2005). They later studied antiapoptotic siRNA against Fas and caspase-8 assessing their capacity to protect the lung from the detrimental effects of HEM-induced septic ALI (Perl M et al. Am. J. Pathol. 167:1545-59, 2005). Using mice overexpressing GFP and receiving a single i.t. instillation of GFP-silencing RNA, they found that green fluorescence in the lungs (18 h post-instillation) was reduced (compared with vehicle-treated GFP mice0, while no decrease in fluorescence was seen in other organs (Perl et al., supra; Lomas-Neira et al., supra). The use of siRNA at these concentrations did not induce an interferon response (via activation of various TLR pathways nor via proinflammatory processes). However, the induction of STAT-1 by this treatment could not be ruled out. The authors' group followed the intrapulmonary deposition of Cy-5 fluorochrome labeled siRNA uptake by confocal immunofluorescence microscopy and found that labeled siRNA co-localized only with lung epithelial cells (“EC”) at 18 at 24 h post instillation, but not with alveolar macrophages (“AM”). The feasibility of gene silencing in macrophages using siRNA has been described in vitro, however, silencing of typically macrophage-derived molecules such as TNF-α and IL-6 during indirect murine lung injury remained unsuccessful. Experiments were designed to modulate PMN immigration based on the “neutrophil hypothesis” using in vivo siRNA constructs against murine chemokines KC and MIP-2 during the development of indirect septic ALI. Suitable siRNA constructs were instilled into lungs 2 h following HEM (and prior to induction of sepsis by cecal ligation and puncture), and ALI was assessed 24 h later. Silencing of MIP-2 reduced tissue and plasma IL-6 concentrations, tissue MIP-2, and lung PMN influx, interstitial edema, alveolar congestion, and disruption of lung tissue architecture (citing to Lomas-Nara et al., supra). In contrast, KC-siRNA treatment, while reducing plasma KC, tissue KC, and tissue IL-6, did not significantly reduce plasma IL-6 nor lung neutrophil influx nor lung damage. siRNA sequences specific for Fas and caspase-8 were instilled i.t. during septic ALI and diminished gene-specific lung Fas and caspase-8 expression; pulmonary tissue caspase-3 activity was reduced only in response to Fas but not caspase-8 silencing. Silencing of Fas in lung ECs was associated with a reduction in lung inflammation and neutrophil influx. It was not known whether different forms of ALI (e.g., direct versus indirect) are more of a response to a certain pathogenic mechanism. While numerous diverse stimuli can initiate the pathogenesis of this clinical entity, the final steps in ALI/ARDS, such as compromise of the alveolo-capillary bather function, appear to be somewhat common. This document reviewed data supporting the independent roles of activated PMN vs EC death in ALI. Upon early Fas activation, AM and lung ECs can produce chemokines in the lung, attracting activated and potentially harmful PMNs, monocytes, or even T cells to the site of injury and potentiating the degree of injury. This documents did not mention or suggest targeting Btk nor using an anti-neutrophil antibody to deliver any siRNA let alone BTK-specific siRNAs to the lungs.
Bojnik et al., Abstract S521, 18th Cong. European Hematology Assoc, 2013, discloses efficient silencing of BTK in primary chronic lymphocytic leukemia (CLL) cells by RNA interference resulting in a 60-90% reduction in protein levels compared to CLL cells ‘nucleofected” with a control siRNA. This resulted in silencing of BTK-accelerated apoptosis. This reference does not disclose conjugating siRNA to any antibody or targeting PMNs in vivo, let alone in the lung or in the treatment ALI.
Peer et al., US Pat. Publ. 2013/0129752 discloses a delivery agent selective for leukocytes or activated leukocytes, comprising a targeting moiety, such as an antibody or functional fragment that selectively binds LFA-1 (integrin Lymphocyte Function-Associated Antigen-1 which is also expressed on neutrophils), a protein carrier moiety covalently linked to the targeting moiety, and a therapeutic agent associated with the carrier moiety—such as a RNA interference molecule (siRNA, dsRNA, stRNA, shRNA, miRNA). Examples of a protein carrier are a basic polypeptide such as protamine or a functional fragment thereof that provide the structure for non-covalent binding to a nucleic acid. This protein carrier serves as a “bridge” between the antibody and the siRNA. Methods for selective delivery to leukocytes/activated leukocytes in vivo, in vitro and ex vivo are disclosed. In the Examples, genetically engineered scFV fragments of two different anti-LFA-1 antibodies were used as the “targeting moiety.” This document does not disclose delivering siRNA to neutrophils in the lung using an antibody construct to treat ALI but it does disclose delivery of siRNA to “lungs” in vivo in an indirect and “model-peculiar” way (para's [0372]-[0374]. This cannot be equated to the present invention's delivery of Btk-siRNA to human lungs in vivo. This protein carrier artifice was needed because human LFA-1 does not cross-react with mouse LFA-1 so that the anti-LFA-1 antibody targeting moiety could not bind to mouse LFA-1 in mouse lungs. Therefore, the authors used immunocompromised SCID mice into which they xenografted human leukemia cells stably transfected to express human LFA-1. After these tumors formed small nodules in mouse lungs, i.v. fusion protein complexes comprising anti-LFA-1 Ab fragments-protamine fusions complexed with siRNA were injected and found their way to the transfected human tumor cells expressing human LFA-1 in the lung and delivered the siRNA. The authors considered this to be in vivo proof of principle for effective systemic siRNA delivery by these fusion proteins to LFA-1-expressing cells. The disclosed compositions and methods required “3-component moieties” that in addition to the targeted therapeutic (siRNA) and the targeting moiety (e.g., antibody) required a carrier protein covalently bonded to the targeting moiety. A basic polypeptide exemplified as a protamine or protamine fragment is contemplated as the carrier protein and acts by binding non-covalently to the siRNA to carry it.
Simon, US2005/0255120 describes a composition comprising: a cell surface receptor specific ligand bonded to a dsDNA that encodes a promoter region. This construct is functionally linked to express siRNA or short hairpin RNA (shRNA) that suppresses production of a cellular protein. Simon, US2005/0260214) discloses a composition comprising: a cell surface receptor-specific immunoglobulin (Ig) (antibody) or Ig component ligand bonded to a dsRNA encoding a siRNA or to a shRNA sequence that suppresses production of a cellular protein. The antibody is specific for a cell surface antigen. These references do not relate to Btk-specific siRNA's or to anti-neutrophil antibodies nor do they disclose or suggest targeting the lung, targeting neutrophils or targeting Btk. (The word “lung” does not appear in these publications.)
Ford et al., WO2009/102782 discloses a conjugate for delivery of a nucleic acid, including siRNA, to cells which comprises a carrier that binds non-covalently to the nucleic acid that is covalently coupled to a ligand that binds to a cell surface, such as an antibody or antigen-binding fragment thereof. This publication describes a method of treating a subject with this conjugate by administering a pharmaceutically acceptable composition comprising a therapeutically effective amount of the conjugate that binds to the nucleic acid non-covalently and at least partially inhibits one or more symptoms of a disease. The document refers to the “promise” of direct delivery of RNAi agents in vivo with affinity reagents, especially antibodies, for treating diseases and disorders characterized by “the under-expression or over-expression of a gene or group of genes, including genes with mutations.” This is said to include metabolic diseases and disorders (e.g., where the liver is a target), infectious diseases caused by viruses, bacteria and fungi, and cancer (particularly myeloid leukemia). Pharmaceutical compositions for pulmonary (aerosol inhalation) or nasal administration, are disclosed. Pulmonary administration is said to include inhalation of aerosolized or nebulized liquid or solid particles of the pharmaceutically active component dispersed in and surrounded by a gas. The only example of in vivo use is injection of a composition that mixes GAPDH-specific siRNA anti-Her-2 mAb conjugated with protamine (which provides the nucleic acid binding capacity) into mice carrying a xenografted human tumor and demonstrating reduction of GAPDH expression in the tumors a few days later. The conjugates described in this document are not conjugates of an antibody with a siRNA (or other nucleic acid) bur rather a 3 part complexes of a conjugate of antibody with a nucleic acid binding molecule (such as a protamine) and a nucleic acid.
Cha et al., WO2012/006083 discloses molecular conjugates of ligands covalently linked, directly or via a linker, to a siRNA moiety and a method for delivering molecular conjugates to a cell. The method comprises (a) contacting a cell or cells with a molecular conjugate comprising a ligand having affinity for a cell surface receptor and an siRNA moiety linked to the ligand; and (b) maintaining the cell, or a population of such cells, under conditions whereby the ligand specifically binds to a cell surface receptor, whereupon the conjugate enters the cells by endocytosis, and delivers the siRNA moiety to the cytoplasm. The document generically discloses non-specific siRNA delivery but notes that cell-type-specific delivery is the most challenging step blocking the progress of RNAi therapy. It notes that targeting siRNA to specific cell or tissue types requires that the specificity be built into the delivery agents or the expressed siRNAs, for example by antibody targeting (citing Yu et al. AAPS J. 11:195-203, 2009, see below). The disclosure focuses on ligands for surface receptors, primarily muscarinic receptors and the targeting of exocrine glandular cells of salivary gland, lacrimal gland, tracheobronchial gland, digestive gland, or sweat gland. Therapeutic focus is on Sjogren's Syndrome and the silencing of inflammatory caspases. The examples are limited to targeting conjugates of carbachol (a muscarinic ligand) with siRNA specific for caspase-3 to human salivary gland cells. While intrapulmonary and intranasal administration appears in a long list of routes of administration, this application does not specifically disclose any lung disease nor its treatment by administering such a conjugate to lung tissue.
Yu, B. et al., AAPS J. 11:195-203, 2009, discloses various conjugates of oligonucleotides such as siRNA with ligands for cell-specific or site-specific delivery of the oligonucleotides. The following examples of target tissues and delivery strategies are disclosed using antibody targeting moieties:
Target tissues/cellsDelivery StrategiesRefActivated leukocytes/K562Antibody-protamine-siRNAs(a)leukemiacomplexesLeukocytesβ7 integrin Ab-HA coated(b)liposomesMammary carcinomaanti-HER2 antibody (61),(c)Human B-lymphoma cell linesCD19-targeted liposomes(d)Neuroblastoma or melanomaGD2 ganglioside-targeted(e)immunoliposomes(a) E. Song, et al. Nat. Biotechnol. 23:709-17, 2005;(b) D. Peer, et al. Proc. Natl. Acad. Sci. U.S.A. 104:4095-4100, 2007);(c) D. B. Kirpotin et al., Cancer Res. 66:6732-40, 2006.;(d) D. D. Stuart et al., Cancer Gene Ther. 7:466-75, 2000;(e) G. Pagnan et al., J. Natl. Cancer Inst. 92:253-61 (2000)This reference does not describe or suggest an oligonucleotide conjugated to neutrophil-specific antibody nor selective delivery by any antibody of an oligonucleotide to any cell in the lungs.